Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model and pilot data.

Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear-nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

Impact of COVID-19 on the private and professional lives of highly educated women working in global health in Europe-A qualitative study.

Background: The COVID-19 pandemic has led to a deepening of existing inequalities and a rollback of achievements made in gender equality. Women in Global Health (WGH) is a global movement that aims to achieve gender equality in health and increase female leadership in global health. Here, the aim was to understand how the pandemic affects the private and professional lives of women working in global health in different European countries. Suggestions for future pandemic preparedness including how gender perspectives should be integrated into pandemic preparedness and how a women's network such as WGH helped them to overcome the impact of the pandemic were explored. Methods: Qualitative semi-structured interviews were conducted in September 2020 with a sample size of nine highly educated women with a mean age of 42.1 years from the different WGH European chapters. The participants were informed of the study and were formally asked for their consent. The interviews were held in English via an online videoconference platform and lasted 20-25 min each. The interviews were audio recorded and transcribed verbatim. Thematic analysis was conducted according to Mayring Qualitative Content Analysis using MAXQDA. Results: The pandemic has both positive and negative effects on the professional and private lives of women. It led to an increased workload and stress as well as pressure to publish on COVID-19-related themes. Increased childcare and household responsibilities represented a double burden. The available space was limited if other family members were also working from home. Positive aspects included more time for family or partners and reduced travel. The participants report on perceived gender differences in the experience of the pandemic. International cooperation is considered to be a key factor for future pandemic preparedness. Being part of a women's network such as WGH was perceived as being very supportive in difficult situations during the pandemic. Conclusion: This study provides unique insights into the experiences of women working in global health in different European countries. The COVID-19 pandemic influences their professional and private lives. Perceived gender differences are reported and suggest the need for integrating gender perspectives in pandemic preparedness. Networks for women, such as WGH, can facilitate the exchange of information in crises and provide women with professional and personal support.

Distribution and Structure of DM2 Repeat Tract Alleles in the German Population.

Autosomal dominant inherited Myotonic dystrophy type 1 and 2 (DM1 and DM2) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. For Germany cumulative empiric evidence suggests an estimated prevalence of DM2 of roughly 9 in 100,000, therefore being as prevalent as DM1. In DM2, a (CCTG)n repeat tract located in the first intron of the CNBP gene is expanded. The CCTG repeat tract is part of a complex repeat structure comprising not only CCTG tetraplets but also repeated TG dinucleotides and TCTG tetraplet elements as well as NCTG interruptions. Here, we provide the distribution of normal sized alleles in the German population, which was found to be highly similar to the Slovak population. Sequencing of 34 unexpanded healthy range alleles in DM2 positive patients (heterozygous for a full expansion) revealed that the CCTG repeat tract is usually interrupted by at least three tetraplets which according to current opinion is supposed to render it stable against expansion. Interestingly, only the largest analyzed normal allele had 23 uninterrupted CCTGs and consequently could represent an instable early premutation allele. In our diagnostic history of DM2 cases, a total of 18 premutations were detected in 16 independent cases. Here, we describe two premutation families, one with an expansion from a premutation allele and the other with a contraction of a full expansion down to a premutation allele. Our diagnostic results support the general assumption that the premutation range of unstable CCTG stretches lies obviously between 25 and 75 CCTGs. However, the clinical significance of premutation alleles is still unclear. In the light of the two described families we suggest incomplete penetrance. Thus, as it was proposed for other repeat expansion diseases (e.g., Huntington's disease), a fluid transition of penetrance is more likely rather than a clear cut CCTG number threshold.

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation-negative early-onset and high-risk breast cancer patients.

To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2-mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age-matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease-causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk.

Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes.

To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4- to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans.

In vitro maturation of oocytes is not associated with altered deoxyribonucleic acid methylation patterns in children from in vitro fertilization or intracytoplasmic sperm injection.

OBJECTIVE: To study the possible transmission, to the next generation, of epigenetic defects associated with in vitro maturation (IVM) of human oocytes. DESIGN: Case-control study using epigenetic data. SETTING: Two collaborating university departments. PATIENT(S): Eleven IVM newborns and 19 controls, conceived by conventional assisted reproduction. INTERVENTION(S): Chorionic villus and cord-blood sampling. MAIN OUTCOME MEASURE(S): Using bisulfite pyrosequencing, we have measured average methylation levels of 6 imprinted (LIT1, MEG, MEST, NESPas, PEG3, and SNRPN), 5 tumor-suppressor (APC, ATM, BRCA1, RAD51C, and TP53), 2 pluripotency (NANOG and OCT4), and 2 metabolic (LEP and NR3C1) genes, as well as 2 repetitive elements (ALU and LINE1) in 2 tissues of IVM and control neonates. Using deep bisulfite sequencing, we have determined methylation patterns of many individual DNA molecules to detect rare RAD51C epimutations (allele methylation errors). RESULT(S): No statistically significant impact was found of IVM on chorionic villus and cord-blood DNA methylation at the studied developmentally important genes and interspersed repeats. The RAD51C epimutation rate was low (0.5% ± 0.1%) in all analyzed samples. CONCLUSION(S): IVM-induced epigenetic changes in offspring, if any, are relatively small in magnitude and/or infrequent.

Stress appraisals and training performance on a complex laboratory task.

OBJECTIVE: We investigated whether training performance on a complex laboratory task differs for trainees whose stress appraisals denote challenge or threat. BACKGROUND: Past research outside a training context found better performance on tasks when stress appraisals denoted challenge (in which perceived situational demands are commensurate with perceived resources) as opposed to threat (in which perceived coping resources fall short of the demands of the stressor). METHOD: College students performed Space Fortress during 80 3-min practice trials, 20 tests, and posttests (retention, transfer, secondary task interference). Stress appraisals were measured with a two-item scale (Experiments 1-3) or an eight-item scale (Experiment 3) with (Study 1) or without (Experiments 2 and 3) brief hands-on experience. RESULTS: In all experiments, training improved performance and challenged trainees outperformed threatened trainees throughout training as well as on some baseline and posttraining tests. CONCLUSIONS: These studies are the first to document that stress appraisals predict training performance. They suggest that little information about a task is needed for appraisals to account for a significant amount of variance (11%) in training performance. Investigating the dynamic interplay of stress appraisals and training will increase the understanding of stress appraisals and of training. APPLICATION: Stress appraisals may improve training if used as a screening tool and/or by implementing interventions aimed at changing appraisals from threat to challenge.